Abstract

Cloning of multiple genes in a single vector has greatly facilitated both basic and translational studies that require co-expression of multiple factors or multi-units of complex protein. Many strategies have been adopted, among which 2A "self-cleaving" peptides have garnered increased interest for their polycistronic nature, small size and high "cleavage" efficiency. However, broad application of 2 A peptides is limited by the lack of systematic comparison of different 2As alone or in combination. Here we characterized the effect of varying gene position and 2As on the expression of proteins encoded in bi-, tri-, or quad-cistronic constructs. Using direct cardiac reprogramming as an example, we further determined the effect of varied 2As on the efficiency of fluorescent cell labeling and cell fate conversion. We found that the expression of fluorophores decreased as it was moved towards the end of the construct while reprogramming was most efficient with the fluorophore at the second position. Moreover, quad-cistronic TPE2A constructs resulted in more efficient reprogramming than 3P2A or PTE2A constructs. We expect that the bi-, tri-, and quad-cistronic vectors constructed here and our results on protein expression ratios from different 2A constructs could serve to guide future utilization of 2A peptides in basic research and clinical applications.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

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Construction of the multi-gene co-expression…

Figure 1

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Construction of the multi-gene co-expression vector with 2A sequences. ( A ) Schematic…

Figure 1

Construction of the multi-gene co-expression vector with 2A sequences. (A) Schematic representation of the mechanism of “self-cleaving” 2A peptides. (B) DNA and amino acid sequences of the various 2As used. A GSG linker was added to the N-terminus of all 2As. (C) A simplified map of the pGEM-T-PTE2A cloning vector showing 2As and restriction sites used.

Figure 2

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Bi-cistronic 2A constructs: impact of…

Figure 2

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Bi-cistronic 2A constructs: impact of 2A sequences on the expression of the gene…

Figure 2

Bi-cistronic 2A constructs: impact of 2A sequences on the expression of the gene at the second position. (A) Different bi-cistronic 2A constructs encoding Mef2c (M) and Gfp. “t” is the abbreviation for “tandem.” (B) Representative live fluorescent images of various cell types transduced (except transfection in 293 T) with retroviruses (pMXs) encoding the different 2A constructs. MEF, mouse embryonic fibroblast. MEF-T, immortalized MEF cell line. CF, primary cardiac fibroblast. TTF, primary tail-tip fibroblast. Boxed area in MEF-T Mock and M-tPTE2A-GFP are longer exposed to show that the percentage of GFP+ cells were 100% for M-tPTE2A-GFP even though GFP intensity is low. Images were taken on day 3 post-transduction or 48 hr post-transfection (293T) at 20X (40X for MEF-T). Scale bar = 100 µm. (C) Cells in (B) were collected and quantified for percentage of GFP+ cells (green triangle) and the fluorescence intensity of GFP (purple diamond) by flow cytometry. dMFI, delta median fluorescence intensity = MFI of GFP+ cells − MFI of GFP− cells. dMFI of all 2A constructs were normalized to that of the GFP-tPT2A-M. (D) Western blots showing Mef2c and GFP protein expression in MEF-T cells transduced with different bi-cistronic constructs. #Spliced protein. <Unspliced M-2A-GFP. Middle panel: a longer exposure was used to visualize the unspliced GFP. Right panel: the expression levels of unspliced Mef2c and GFP were quantified and normalized to the β-actin loading control and then GFP/Mef2c ratios in different M-2A-GFP constructs were calculated and further normalized to that in the M-P2A-GFP construct. Mean ± SD of triplicate experiments were shown. Statistical significance was calculated by one-way ANOVA and bonferroni correction.

Tri-cistronic 2A constructs: positional effects on gene expression. (A) Tri-cistronic constructs in a P2A-T2A backbone expressing three FPs GFP, Td and i670 in different orders. (B and C) MEF-T cells were transduced with retroviruses (pMXs) encoding the different 2A constructs for 3 days. Live fluorescent images of these cells were taken at 20X (B) and then cells were collected for (C) western blotting analysis for GFP expression, or (D) flow cytometry analysis for dMFI of GFP (green bar), Td (red bar), and i670 (purple bar). (C) GFP was quantified and normalized to the β-actin loading control. (D) dMFI was normalized to that of GFP-Td-i670 for GFP, Td-i670-GFP for Td, and i670-Td-GFP for i670. (E) The same as (D) except performed in TTF. Mean ± SEM of multiple experiments were shown. Results from one-way ANOVA and bonferroni correction were summarized in a table. Scale bar = 200 µm.

Quad-cistronic 2A constructs: example of application in cellular reprogramming. (A) Quad-cistronic constructs using different orders of 2As (PTE2A, TPE2A, and 3P2A) for the expression of Mef2c (M), Gata4 (G), Tbx5 (T) and Tdtomato (Td). A total of 12 constructs were built with Td rotating from position 1 to 4. (B) Flow cytometry quantification of the percentage of Td+ cells (red triangle) and dMFI of Td (purple diamond) in various cell types transduced (except transfection in 293T) with retroviruses (pMXs) encoding the different 2A constructs. dMFI of Td was normalized to that in the PTE2A-TdMGT construct. Cells were collected on day 3 post-transduction or 48 hr post-transfection (293T). (C) Western blots for M, G, T expression in PTE2A and TPE2A constructs. Right panel: quantification after normalization to the β-actin loading control. (D) Positional effects on protein expression in quad-cistronic constructs calculated based on (C). (E–G) Td expression and reprogramming with different 2A constructs in MEF-T on day 3 post-transduction. (E) Representative live fluorescent images of Td and the reprogramming reporter αMHC-GFP. All taken at 20X. Scale bar = 200 µm. (F) Flow cytometry quantification of Td and GFP single- and double-positive cells. (G) GFP dMFI in αMHC-GFP+ cells (blue bar) and the product of the percentage of αMHC-GFP+ cells and dMFI-GFP (green bar). dMFI of GFP was normalized to that of the TPE2A-MGTTd construct. Mean ± SD of triplicate experiments were shown in (B,D,E).